植物与病原菌互作的蛋白质组学研究进展

蛋白质组学作为功能基因组学研究的主要内容之一,在阐述基因功能、了解生命现象和本质的分子机制等方面发挥着重要作用。植物蛋白质组学作为蛋白质组学的一个分支,研究应用也越来越广泛,尤其是探索植物与病原菌互作机制是其中的一个研究热点。本文就多年来植物与真菌、病毒、细菌互作的蛋白质组学研究做一综述,并对当前该领域今后的研究方向进行展望,以期为相关研究提供一些参考和理论基础。     

Shooting yourself in the foot: How immune cells induce antibiotic tolerance in microbial pathogens

Antibiotic treatment failure of infection is common and frequently occurs in the absence of genetically encoded antibiotic resistance mechanisms. In such scenarios, the ability of bacteria to enter a phenotypic state that renders them tolerant to the killing activity of multiple antibiotic classes is thought to contribute to antibiotic failure. Phagocytic cells, which specialize in engulfing and destroying invading pathogens, may paradoxically contribute to antibiotic tolerance and treatment failure. Macrophages act as reservoirs for some pathogens and impede penetration of certain classes of antibiotics. In addition, increasing evidence suggests that subpopulations of bacteria can survive inside these cells and are coerced into an antibiotic-tolerant state by host cell activity. Uncovering the mechanisms that drive immune-mediated antibiotic tolerance may present novel strategies to improving antibiotic therapy.     

水平基因转移介导抗菌素耐药性传播机制的研究进展

近几十年来,病原菌耐药性的出现和蔓延已上升为严峻的公共卫生问题。越来越多研究表明,抗菌素抗性基因(antibiotic resistance genes,ARGs)不仅仅见于临床所分离的病原体,而是包括所有的致病菌、共生菌以及环境中的细菌,它们都能在可移动遗传元件和噬菌体的作用下,通过水平基因转移(horizontal gene transfer,HGT)途径获得耐药性,进而形成抗菌素耐药基因簇(耐药基因组)。HGT可导致抗菌素的耐药性在环境共生菌和病原菌之间传播扩散,这可通过临床上一些重要的抗菌素耐药基因的传播证实。传统观念认为HGT的三种机制中,接合对ARGs的传播影响最大,最近研究表明转化和转导对ARGs播散起到不可忽视的作用。通过深入了解耐药基因组的传播及其在动员病原菌耐药中发挥的作用,对于控制这些基因的播散是至关重要的。将讨论耐药基因组的概念,提供临床相关的抗菌素抗性基因水平基因转移的例子,对当前已研究的促使抗菌素耐药性传播的各种HGT机制进行回顾。     

Proteomics for identifying mechanisms and biomarkers of drug resistance in cancer

A major problem in chemotherapy of cancer patients is drug resistance as well as unpredictable response to treatment. During chemotherapy, multiple alterations of genetics and epigenetics that contribute to chemoresistance take place, eventually impacting on disease outcome. A more complex picture of the mechanisms of drug resistance is now emerging through application of high-throughput proteomics technology. We have entered an exciting time where proteomics are being applied to characterize the mechanisms of drug resistance, and to identify biomarkers for predicting response to chemotherapy, thereby leading to personalized therapeutic strategies of cancer patients. Comparative proteomics have identified a large number of differentially expressed proteins associated with chemoresistance. Although roles and mechanisms of such proteins in chemoresistance need to be further proved, at least some of them may be potential biomarkers for predicting chemotherapeutic response. Herein, we review the recent advancements on proteomic investigation of chemoresistance in human cancer, and emphasize putative biomarkers for predicting chemotherapeutic response and possible mechanisms of chemoresistance identified through proteomic approaches. Suggested avenues for future work are discussed.     

Crossroads of Antibiotic Resistance and Biosynthesis

The biosynthesis of antibiotics and self-protection mechanisms employed by antibiotic producers are an integral part of the growing antibiotic resistance threat. The origins of clinically relevant antibiotic resistance genes found in human pathogens have been traced to ancient microbial producers of antibiotics in natural environments. Widespread and frequent antibiotic use amplifies environmental pools of antibiotic resistance genes and increases the likelihood for the selection of a resistance event in human pathogens. This perspective will provide an overview of the origins of antibiotic resistance to highlight the crossroads of antibiotic biosynthesis and producer self-protection that result in clinically relevant resistance mechanisms. Some case studies of synergistic antibiotic combinations, adjuvants, and hybrid antibiotics will also be presented to show how native antibiotic producers manage the emergence of antibiotic resistance.     

植物与病原菌互作的蛋白质组学研究进展

深入认识植物与病原菌的识别方式、亲和性或非亲和性的互作模式,对于揭示植物-病原菌互作机制研究具有重要意义.利用蛋白质组学方法研究病原菌侵染植物过程,分析相关的基因和蛋白,有助于从分子水平上探究植物-病原菌相互作用机制.本文概述了植物-病原菌的互作机制,系统介绍了差异蛋白质组学分析方法在植物-病原真菌、植物-病原细菌两类互作系统中的应用,分析了植物与病原菌互作过程中可能涉及的差异表达功能蛋白,并对当前蛋白质组学技术在植物与病原菌互作研究中存在的诸多问题进行了探讨.     

Aminoacyl-tRNA synthetases and their inhibitors as a novel family of antibiotics

The emergence of multidrug-resistant strains of pathogenic microorganisms and the slow progress in new antibiotic development has led in recent years to a resurgence of infectious diseases that threaten the well-being of humans. The result of many microorganisms becoming immune to major antibiotics means that fighting off infection by these pathogens is more difficult. The best strategy to get around drug resistance is to discover new drug targets, taking advantage of the abundant information that was recently obtained from genomic and proteomic research, and explore them for drug development. In this regard, aminoacyl-tRNA synthetases (ARSs) provide a promising platform to develop novel antibiotics that show no cross-resistance to other classical antibiotics. During the last few years there has been a comprehensive attempt to find the compounds that can specifically target ARSs and inhibit bacterial growth. In this review, the current status in the development of ARS inhibitors will be briefly summarized, based on their chemical structures and working mechanisms.     

Advances in shotgun proteomics and the analysis of membrane proteomes

The emergence of shotgun proteomics has facilitated the numerous biological discoveries made by proteomic studies. However, comprehensive proteomic analysis remains challenging and shotgun proteomics is a continually changing field. This review details the recent developments in shotgun proteomics and describes emerging technologies that will influence shotgun proteomics going forward. In addition, proteomic studies of integral membrane proteins remain challenging due to the hydrophobic nature in integral membrane proteins and their general low abundance levels. However, there have been many strategies developed for enriching, isolating and separating membrane proteins for proteomic analysis that have moved this field forward. In summary, while shotgun proteomics is a widely used and mature technology, the continued pace of improvements in mass spectrometry and proteomic technology and methods indicate that future studies will have an even greater impact on biological discovery.     

Proteome analysis of host-pathogen interactions: Investigation of pathogen responses to the host cell environment

Infectious diseases are still a major health risk, and thus a better understanding of the interaction between human host cells and pathogenic microbes is urgently required. Since the interplay between both partners is highly complex, genome-wide analysis by OMICs approaches will likely make a major contribution to the elucidation of the pathophysiology of infection processes. In the concert of OMICs technologies, proteomics is particularly important because it reveals changes in the active players of the cell and has thus a close relationship to the phenotypic changes observed. While proteomic studies of in vitro-grown microbial pathogens are routinely established in many labs, in vivo proteomic approaches are still rare. Here, we will review the challenges and recent developments of proteomic analysis of microbial pathogens derived from cell culture or in vivo infection settings and summarize some lessons that have been learned from these studies.     

Label-free proteomics and systems biology analysis of mycobacterial phagosomes in dendritic cells and macrophages

Proteomics has been applied to study intracellular bacteria and phagocytic vacuoles in different host cell lines, especially macrophages (Mφs). For mycobacterial phagosomes, few studies have identified over several hundred proteins for systems assessment of the phagosome maturation and antigen presentation pathways. More importantly, there has been a scarcity in publication on proteomic characterization of mycobacterial phagosomes in dendritic cells (DCs). In this work, we report a global proteomic analysis of Mφ and DC phagosomes infected with a virulent, an attenuated, and a vaccine strain of mycobacteria. We used label-free quantitative proteomics and bioinformatics tools to decipher the regulation of phagosome maturation and antigen presentation pathways in Mφs and DCs. We found that the phagosomal antigen presentation pathways are repressed more in DCs than in Mφs. The results suggest that virulent mycobacteria might co-opt the host immune system to stimulate granuloma formation for persistence while minimizing the antimicrobial immune response to enhance mycobacterial survival. The studies on phagosomal proteomes have also shown promise in discovering new antigen presentation mechanisms that a professional antigen presentation cell might use to overcome the mycobacterial blockade of conventional antigen presentation pathways.     

Proteomics of industrial fungi: trends and insights for biotechnology

Filamentous fungi are widely known for their industrial applications, namely, the production of food-processing enzymes and metabolites such as antibiotics and organic acids. In the past decade, the full genome sequencing of filamentous fungi increased the potential to predict encoded proteins enormously, namely, hydrolytic enzymes or proteins involved in the biosynthesis of metabolites of interest. The integration of genome sequence information with possible phenotypes requires, however, the knowledge of all the proteins in the cell in a system-wise manner, given by proteomics. This review summarises the progress of proteomics and its importance for the study of biotechnological processes in filamentous fungi. A major step forward in proteomics was to couple protein separation with high-resolution mass spectrometry, allowing accurate protein quantification. Despite the fact that most fungal proteomic studies have been focused on proteins from mycelial extracts, many proteins are related to processes which are compartmentalised in the fungal cell, e.g. β-lactam antibiotic production in the microbody. For the study of such processes, a targeted approach is required, e.g. by organelle proteomics. Typical workflows for sample preparation in fungal organelle proteomics are discussed, including homogenisation and sub-cellular fractionation. Finally, examples are presented of fungal organelle proteomic studies, which have enlarged the knowledge on areas of interest to biotechnology, such as protein secretion, energy production or antibiotic biosynthesis.     

Contributions of proteomics to understanding phagosome maturation

In metazoans macrophage cells use phagocytosis, the process of engulfing large particles, to control the spread of pathogens in the body, to clear dead or dying cells, and to aid in tissue remodelling, while the same process is also used by unicellular eukaryotes to ingest food. Phagocytosing cells essentially swallow the particles, trapping them in vacuoles called phagosomes that go through a series of maturation steps, culminating in the destruction of the internalized cargo. Because of their central role in innate immunity and their relatively simple structure (one membrane bilayer surrounding a single particle), phagosomes have been a popular subject for organelle proteomics studies. Qualitative proteomic technologies are now very sensitive so hundreds of different proteins have been identified in phagosomes from several species, revealing new properties of these intriguing compartments. More recently, quantitative proteomic approaches have also been applied, shedding new light on the dynamics and composition of maturing phagosomes. In this review we summarize the studies that have applied proteomic technologies to phagosomes and how they have changed our understanding of phagosome biology.     

Investigation of rifampicin resistance mechanisms in Brucella abortus using MS-driven comparative proteomics

Mutations in the rpoB gene have already been shown to contribute to rifampicin resistance in many bacterial strains including Brucella species. Resistance against this antibiotic easily occurs and resistant strains have already been detected in human samples. We here present the first research project that combines proteomic, genomic, and microbiological analysis to investigate rifampicin resistance in an in vitro developed rifampicin resistant strain of Brucella abortus 2308. In silico analysis of the rpoB gene was performed and several antibiotics used in the therapy of Brucellosis were used for cross resistance testing. The proteomic profiles were examined and compared using MS-driven comparative proteomics. The resistant strain contained an already described mutation in the rpoB gene, V154F. A correlation between rifampicin resistance and reduced susceptibility on trimethoprim/sulfamethoxazole was detected by E-test and supported by the proteomics results. Using 12?836 MS/MS spectra we identified 6753 peptides corresponding to 456 proteins. The resistant strain presented 39 differentially regulated proteins most of which are involved in various metabolic pathways. Results from our research suggest that rifampicin resistance in Brucella mostly involves mutations in the rpoB gene, excitation of several metabolic processes, and perhaps the use of the already existing secretion mechanisms at a more efficient level.     

Advances in quantitative proteomics via stable isotope tagging and mass spectrometry

The high-throughput identification and accurate quantification of proteins are essential components of proteomic strategies for studying cellular functions and processes. Techniques that are largely based on stable isotope protein or peptide labeling and automated tandem mass spectrometry are increasingly being applied in quantitative proteomic studies. Over the past year, significant progress has been made toward improving and diversifying these technologies with respect to the methods for stable isotope labeling, process automation and data processing and analysis. Advances in stable isotope protein labeling and recent biological studies that used stable isotope based quantitative proteomics techniques are reviewed.     

Emerging High-Level Tigecycline Resistance: Novel Tetracycline Destructases Spread via the Mobile Tet(X)

Antibiotic resistance in bacteria has become a great threat to global public health. Tigecycline is a next-generation tetracycline that is the final line of defense against severe infections by pan-drug-resistant bacterial pathogens. Unfortunately, this last-resort antibiotic has been challenged by the recent emergence of the mobile Tet(X) orthologs that can confer high-level tigecycline resistance. As it is reviewed here, these novel tetracycline destructases represent a growing threat to the next-generation tetracyclines, and a basic framework for understanding the molecular epidemiology and resistance mechanisms of them is presented. However, further large-scale epidemiological and functional studies are urgently needed to better understand the prevalence and dissemination of these newly discovered Tet(X) orthologs among Gram-negative bacteria in both human and veterinary medicine.     

Identification of potentially involved proteins in levofloxacin resistance mechanisms in Coxiella burnetii

The etiological agent of Q fever, Coxiella burnetii, is an obligate intracellular bacterium that multiplies within a phagosome-like parasitophorous vacuole. Fluoroquinolones have been used as an alternative therapy for Q fever. Resistance to fluoroquinolones can arise via several mechanisms utilized by pathogens to avoid killing. Until today, genome-based studies have shown that the main mechanism of C. burnetii to resist inhibition by fluoroquinolones is based on mutations in quinolone-resistance-determining region (QRDR). In this study, in a broader search at the protein level for C. burnetii mechanisms that confer resistance to fluoroquinolones, the proteomes of in vitro developed fluoroquinolone resistant bacteria and susceptible bacteria were compared using the MS-driven combined fractional diagonal chromatography (COFRADIC) proteomics technique. Quantitative comparison of the 381 proteins identified in both strains indicated the different expression of 15 bacterial proteins. These proteins are involved in different cellular processes indicating that the antibiotic resistance mechanism of the bacterium is a multifaceted process.     

Microbial environments confound antibiotic efficacy

The increasing prevalence of bacteria that are insensitive to our current antibiotics emphasizes the need for new antimicrobial therapies. Conventional approaches to antibacterial development that are based on the inhibition of essential processes seem to have reached the point of diminishing returns. The discovery that diverse antibiotics stimulate a common oxidative cell-death pathway represents a fundamental shift in our understanding of bactericidal antibiotic modes of action. A number of studies, as discussed above, also provide hints about how intra- and extracellular metabolism can enable antibiotic resistance and tolerance. We have, nonetheless, just begun to understand the repertoire of tactics that bacteria use to evade antibiotics. Biosynthetic pathways for natural antibiotics are ancient, and numerous mechanisms for antibiotic resistance and tolerance are likely to have evolved over the past few million years. Unraveling these mechanisms will require concerted efforts by chemical biologists, microbiologists and clinicians. These efforts will benefit from the use of metabolic models and other network-biology approaches to guide investigation of processes that modulate antibiotic susceptibility. Importantly, by helping to identify common points of vulnerability as well as key differences between pathogens, these models may lead to the development of effective adjuvants, novel antibiotics and new antimicrobial strategies. There is also a crucial need to better understand how bacteria within a population cooperate to overcome antibiotic treatments. Such investigations may benefit from the use of novel chemical probes and experimental techniques to interrogate the physiology and functional dynamics of natural microbial communities. Insights gained from these studies will augment metagenomic models that can be used to identify biomolecules responsible for these cooperative strategies. Leveraging chemical biology methodologies and systems-biology approaches for further studies of microbial environments may reveal a wealth of untapped targets for the development of novel compounds to counter the growing threat of resistant and tolerant bacterial infections.     

Proteomics meets blood banking: Identification of protein targets for the improvement of platelet quality

Proteomics has brought new perspectives to the fields of hematology and transfusion medicine in the last decade. The steady improvement of proteomic technology is propelling novel discoveries of molecular mechanisms by studying protein expression, post-translational modifications and protein interactions. This review article focuses on the application of proteomics to the identification of molecular mechanisms leading to the deterioration of blood platelets during storage — a critical aspect in the provision of platelet transfusion products. Several proteomic approaches have been employed to analyse changes in the platelet protein profile during storage and the obtained data now need to be translated into platelet biochemistry in order to connect the results to platelet function. Targeted biochemical applications then allow the identification of points for intervention in signal transduction pathways. Once validated and placed in a transfusion context, these data will provide further understanding of the underlying molecular mechanisms leading to platelet storage lesion. Future aspects of proteomics in blood banking will aim to make use of protein markers identified for platelet storage lesion development to monitor proteome changes when alterations such as the use of additive solutions or pathogen reduction strategies are put in place in order to improve platelet quality for patients.